Production of pharmaceuticals, clinical diagnostics and industrial materials using genetic engineering technique has greatly contributed to the actual industrial world already, among which substance production systems are particularly widely utilized where cultured cells of microorganisms or mammals are used as host cells. However, culture of these cells requires culture facilities or culture media in completed sterile environment. Further inevitable consumption of petroleum energy causes high cost. In addition, mammal cells cannot be used as hosts without involving the risk of contamination of virus which is harmful to human body.
Consequently, substance production systems using transformed plants have been developed as substance production systems with safety at low cost in stead of substance production systems by culture of cells of microorganisms or mammals. For instance, generation of transformed plants producing: a polymeric compound such as biodegradable polyester (e.g. Japanese Laid-Open Patent Application No.2002-262886), a protein such as a vaccine (e.g. G. Jaeger et al., Eur. J. Biochem. 259, 426, 1999) and lactoferrin (D. Chong et al., Transgenic. Res. 9, 71, 2000), and a peptide such as enkephalin (Japanese-Laid Open Patent Application No. 2000-106890), has been reported so far.
With regard to transformed plants, production of a functional substance being beneficial to human body in edible parts of the plants e.g. seeds of Glycine max or Oryza sativa, or vegetable leaves, allows the intended substance to be taken orally into human body directly without an extraction process for them. Further, for seeds, refrigeration or transported in facility with refrigerating device is not required, while it can be steadily stored for long time at room temperature. In addition, even when the intended substance is extracted, it can be easily purified, because, unlike leaves, the contamination of phenolic substances seldom occurs with seeds. Accordingly, a seed has been regarded as an ideal organ to produce the intended genetic product, and generation of seeds which produced: proteins such as glycinin (T. Katsube et al., Plant. Physiol. 120, 1063, 1999), enzymes such as (1,3-1,4)-β-glucanase (H. Horvathetal., Proc. Nathl. Acad. Sci. USA., 97,1914, 2000), and peptides such as enkephalin (D. Chong et al., Transgenic. Res., 9, 71, 2000) has been reported so far.
However, the substance production systems by transformed plants have above superior properties, whereas their production efficiency is inferior to that of culture systems of microorganisms or mammal cells which are the current mainstream, particularly, the production efficiency by plant storage organs was low. In order to solve this problem, measures are variously being devised to enhance the ability of producing substances in transformed plants. For instance, in order to improve the ability of producing substances in one of the storage organ, i.e. a seed, from the point of view to enhance the expression of the introduced intended gene and accumulation of a gene product, studies regarding: utilization of a promoter of a plant storage protein expressed intensively in seeds (e.g. T. Katube et al., Plant. Physiol., 120, 1063, 1999), concomitant use of this promoter and a transcription factor which acts on the promoter to enhance expression (e.g. D. Yang et al., Proc, Nathl. Acad. Sci. USA., 98, 11438, 2001), insertion of 5′ end untranslated region (e.g. Japanese Laid-Open Patent Application No. 2002-58492), optimization of C+G content in a gene (H. Horvath et al., Proc. Nathl. Acad. Sci. USA., 97, 1914, 2000), addition of tranduction signals to an endoplasmic reticulum (Japanese Laid-Open Patent Application No. 2000-504567), and so on have been performed energetically. It is also reported that the production amount of a foreign gene product in the seed was increased by using a mutant being deficient in a seed storage protein as a plant into which a foreign gene is introduced (Japanese Laid-Open Patent Application No. 2002-58492). However, these improvements have not provided enough substance production ability in seeds, so that development of a novel procedure has been longed.
On the other hand, a GLP-1 (glucagon-like peptide-1) is known as a hormone which is secreted from a digestive tract by food intake and acts on the pancreas to stimulate glucose-dependent insulin secretion. In Type 2 diabetic patients, it is reported that responsiveness to this GLP-1 is maintained, while the production of GLP-1 is impaired. It is expected that development of a GLP-1 agent will lead to the application of the agent to a therapeutic agent for diabetes as an insulin secretion promoter to compensate the lack of the GLP-1. However, the active substance of the GLP-1 is a polypeptide of the GLP-1 (7-36) amide or the GLP-1 (7-37), which are digested and degraded by a digestive enzyme in the gastrointestinal tract and is not absorbed sufficiently, when the GLP-1 is taken orally. Therefore, in the present state, intravenous injection and subcutaneous injection are attempted in clinical practice. Moreover, it is also reported that: the GLP-1 is also subjected to degradation by a dipeptidylpeptidase IV (DPP-IV) which exists in blood and tissues, so the active half-life time of the GLP-1 is so short as 1-2 min, and GLP-1 is easily excreted from the kidney, so its half-life time in blood is within 5 min, all of which prevents the GLP-1 from clinical application.
Hence, a GLP-1 derivative with a long half-life which is not easily degraded has been developed. For instance, followings are included: the 8th position of amino acid substituted derivative (diabetologia 41, 271-278, 1998, Biochem 40, 2860-2869, 2001), an amino acid modulator at N— and C-terminals (WO9808871 etc.), a derivative in which Arg is substituted at its 34th position and its 26th position of Lys is introduced with lipophilic group (WO0007617), and a derivative by amino acid substitution covering all over the sequence (WO9943705 and WO9111457). Further, development of a sustained-release injection preparation which is subcutaneously absorbed slowly, or development of an injection preparation with synthetic Exendin-4 having a GLP-1 like agonist activity and derived from lizard whose half-life time in blood is long, have been performed. However, as they are injection preparations, considering the burden to patients, a novel GLP-1 derivative administered via an alternative route other than injection has been longed.
The object of the present invention is to provide a method for producing a plant storage organ in which a recombinant protein is highly produced, a plant storage organ in which the recombinant protein produced by the method is highly produced, and a novel derivative of a human glucagon-like peptide-1 (GLP-1) which is peptidase-resistant and the use thereof.
In order to enhance substance production in a storage organ of a transformed plant, various attempts have been performed as described above. However, in order for a plant storage organ to function in vivo sufficiently by taking it in which the recombinant protein being useful as pharmaceuticals is produced as food, it is necessary to develop a method for producing a plant storage organ in which the recombinant protein is more highly produced. In the meantime, when the recombinant protein is extracted from plants and processed as pharmaceuticals or functional food, it is important that the recombinant protein is highly produced in these storage organs on the cost front. Therefore, one of the objects of the present invention is to provide a novel method for producing a storage organ in which the recombinant protein is highly produced in transformed plants.
Meanwhile, when a GLP-1 is selected as a recombinant protein which is highly produced in a plant storage organ by said method, a therapeutic effect for diabetes can be expected by merely taking fruits, rice, and so on as normal diet. However, as mentioned above, since this native GLP-1 is digested and degraded by the digestive enzyme in the gastrointestinal tract, it can not be orally administered stably, there is no efficient method for administration except injection in the current status. It can be thought that if the GLP-1 can be passed through stomach without being digested using some method, it is absorbed in the small intestine. However, the GLP-1 must exist as a simple substance when it is absorbed. In that time, a native GLP-1 would lose activity by degradation by an enzyme such as trypsin.
Moreover, as the native GLP-1 is continuously degraded by dipeptidylpeptidase IV even after absorption, a sustained effect cannot be expected. Accordingly, in order to obtain a pharmaceutical effect from oral administration of the GLP-1, it is necessary to design a GLP-1 derivative which is not easily degraded with trypsin or dipeptidylpeptidase IV by amino acid substitution and has the sustained activity.
Therefore, one of the other objects of the present invention is to provide a novel GLP-1 derivative which is resistant to a digestive enzyme such as trypsin and can be administered orally, more preferably, a novel GLP-1 derivative which is resistant to dipeptidylpeptidase IV as well. To accomplish this object, it is required to obtain a GLP-1 derivative which is absorbed when taken as food, and which shows a pharmaceutical effect.